Figures

Fig. 1HNH conformational dynamics reveal a distinct I state as a function of PAM-distal mismatches.

(A) Model shows HNH labeling sites under different conformations of Cas9, using sgRNA-bound (4ZT0) and dsDNA-bound (5F9R) structures. The cysteine-light Cas9 construct is labeled with Cy3 and Cy5 at S867C and S355C positions. (B) Top: Cas9 was incubated with 55-bp-long dsDNA substrates that include PAM and target sequences. Mismatches were introduced at the PAM-distal site. Bottom: DNA binding to Cas9 results in HNH interconversion, determined by a transition from a low to high FRET state. Scissors show the DNA cleavage sites. (C) Steady-state smFRET histograms for Cas9 in the absence and presence of 200 nM DNA targets. A multi-Gaussian fitting (black curve) reveals D, I, and R states of HNH. (D) Representative time traces (top), transition density plots (TDPs; middle), and rates of the major transitions in TDPs (bottom) for various DNA substrates. a.u., arbitrary units.

(A) Schematic for observation of Cas9 conformational dynamics upon landing onto surface-immobilized DNA. (B) Left: A representative smFRET trajectory recorded at 100 Hz shows a brief visit to the I state between initial on-target binding and transitioning into the D state. Right: Single exponential fit (red curve) to the I state dwell time histogram reveals its lifetime (τ, ±95% confidence interval). (C and E) A representative smFRET trajectory at 10 Hz of Cas9 after landing to an on-target and 1–3 bp mm DNA. t = 0 s and dashed vertical lines represent time of landing and acceptor photobleaching, respectively. (D and F) Time-dependent changes in the conformational distribution of Cas9 after DNA landing. (G) Cumulative distribution of first transition to the D state after DNA landing. Red curves show fit to a single exponential function (±95% confidence interval).

(A) smFRET histograms of Cas9 bound to on-target dsDNA in the absence and presence of a divalent cation. (B) Top: The on-target DNA was truncated at the 5′ end of the NTS one base after the target sequence (pdDNA1) and four bases after PAM (pdDNA2). Bottom: smFRET histograms of Cas9 bound to pdDNAs in the absence and presence of a divalent cation. (C) Cleavage of pdDNA1 was initiated by replacing EDTA with 5 mM Mg2+ and monitored by dissociation of the Cy5-labeled NTS 5′ end from Cas9. (D) Still images of Cy5-pdDNA1 bound to surface-immobilized Cas9 after Mg2+ addition (t = 0 s). (E) Percentage of Cy5 spots remain at the surface after Mg2+ flow. Red curves represent fit to single exponential decay (mean ± 95% confidence interval).